Mitogen-activated protein kinases (MAPKs) are serine-threonine protein kinases that are activated by diverse stimuli. MAPKs contribute to the control of cell and tissue homeostasis, cell division and cell death. The MAPK regulatory module includes three kinases that establish a sequential activation pathway comprising a MAPK kinase kinase (MKKK), MAPK kinase (MKK) and MAPK. Currently, mammalian MAPKs comprise five families: ERKI/2, p38 c_, 13, y, _5, JNK1,2,3, ERK3/4 and ERK5. Each family of MAPKs has distinct, but sometimes overlapping, functions in terms of substrate recognition and control of cellular pathways. The hypothesis of this proposal is that two related MAPK kinase kinases, MEKK2 and MEKK3, regulate the MEK5-ERK5 module. In addition, MEKK2 regulates JNK and MEKK3 regulates p38. MEKK2, by binding the adaptor protein LAD, selectively couples to receptor tyrosine kinases and Src. MEKK3 binds a structurally unique adapter protein we have named osmosensing scaffold for MEKK3 (OSM). OSM in response to osmolarity changes translocates MEKK3 from the cytoplasm to plasma membrane ruffles and sites of membrane actin polymerization. Recruitment of OSM/MEKK3 to the plasma membrane is associated with MEKK3 activation. OSM is the first identified adaptor protein in mammalian cells for the activation of MAPK pathways in response to osmolarity changes. Targeted gene disruption of MEKK3, that binds OSM, is embryonic lethal, demonstrating the importance of this pathway in cell physiology. The aims of this proposal include: 1) Define the function of OSM in osmosensing for the regulation MAPK pathways 2) Define the function of the PB 1 domain for interaction of MEKK2 and MEKK3 with MEK5. 3) Generate a conditional MEKK3 knockout so the role of OSM/MEKK3 signaling in adult mice and differentiated cell types can be defined. These studies will define the mechanism for OSM/MEKK3 regulation of MAPK activation in osmoregulation, a critical regulatory response for homeostasis.